Despite the high costs involved, the number of proton therapy centers continues to increase exponentially. Remarkably, even as new centers come on line, there is increasing realization that there are significant gaps in our knowledge of the biologic effectiveness of protons, which limit the clinical potential of proton therapy. One common theme in the literature is that relative biologic effectiveness (RBE) varies substantially as a function of depth of penetration and dose. Yet, in current practice RBE is simplistically assumed to be 1.1 in all situations while computing radiation dose for treatments. This assumption may lead to an increased risk of injury to surrounding normal tissues where RBE may be higher than 1.1. Moreover, the opportunity to take advantage of the higher RBE to achieve greater killing of tumor cells is not realized. To date virtually all clinical proton treatments and biologic measurements of RBE have employed passive scattering proton therapy (PSPT). This has likely masked the importance of RBE variability. Intensity-modulated proton therapy (IMPT) delivered with pristine scanned proton beams is considered to be the future of proton therapy. IMPT is much more versatile than currently prevalent PSPT. However, for true multi-field optimized IMPT the high inhomogeneity of physical dose contributed by individual beams may be substantially affected by RBE variability and go unrecognized even by experienced practitioners. On the other hand, given sufficient knowledge, the inherent flexibility of IMPT planning may allow for the incorporation of RBE spatial variation into the treatment planning process, potentially increasing biologically effective target dose while simultaneously decreasing normal tissue exposure to high RBE regions of each beamlet. The long-term goals of our research are to improve our understanding of the biological effectiveness of protons and to employ the knowledge thus acquired to enhance the efficacy of intensity modulated proton therapy. In order to accomplish RBE optimized IMPT and thereby expand the therapeutic index of proton therapy, detailed spatial data concerning RBE is desperately needed in order to guide the inverse treatment planning process. In the current proposal we will use mono-energetic scanned proton beams, an innovative experimental design based on the physics of radiation transport and high- throughput biological techniques. The following aims will provide data essential for achieving our long-term goals; (1) Enhance a recently developed system for systematically and accurately mapping the biologic effectiveness of particle therapy, (2) Improve our understanding of the variability of RBE based on high- resolution, high accuracy biologic data, (3) Investigate the potential biologic and clinical consequences of spatial RBE variability. In contrast to other studies focusing solely on characterizing RBE using traditional methods, the significance of this proposal lies in generating highly accurate RBE data with unprecedented spatial resolution. Such data will allow for the incorporation of variable biologic effectiveness ito IMPT treatment planning and dramatically expand the therapeutic ratio of particle therapy.